The use of pigs as organ donors for human transplantation represents a solution to the escalating shortage of organs that are available for patients with end-stage diseases. Wild type pig organs are hyperacutely rejected, however, by pre-existing natural antibodies directed at the 1-D-galactosyl-(1->3)-1-D-galactoside (gal 11,3gal) carbohydrate, which is expressed on pig, cells and absent in humans. Organs derived from gal knockout pigs have been bred as potential donors, but recent studies from several laboratories have shown that although these organs do not undergo hyperacute rejection, they are still rejected within six months. Acute humoral rejection (AHXR) and thrombosis contribute to the demise of these grafts. Anti-non-gal xenoantibodies have been shown to be induced in non-human primates transplanted with gal knockout donor organs, but the specificity, origin and structure of these antibodies has not been determined. Currently available immunosuppressive drugs are ineffective in preventing AHXR. Additional research is needed to determine why acute humoral xenograft rejection occurs, and to define a means to prevent it. Our specific aims propose a detailed and systematic study on the origin and binding specificity of xenoantibodies that still reject genetically-manipulated xenografts in non-human primates transplanted with genetically-modified organs. This work would provide information that is lacking but necessary for the design of a successful approach to preventing xenograft rejection. The experimental samples that we plan to examine in our study have been bred on a gal knockout background and have additional transgenes such as CD39 to prevent thrombosis. We will further determine whether an anti-idiotypic antibody that identifies B cells producing xenoantibodies will prevent AHXR in vivo. This grant proposal will extend our work in the previous grant period in which we defined the structure of immunoglobulin genes encoding xenoantibodies to wild type pig organs in non-human primates and have identified an anti-idiotypic antibody with the ability to define B cells producing xenoantibodes. PUBLIC HEALTH RELEVANCE: The use of specifically bred pigs as organ donors for human transplantation (xenotransplantation) could provide organs for the many patients who may otherwise die waiting. These organs function for three to six months before the grafts are rejected by antibodies. This study will identify the genes that encode these antibodies and will test novel ways to prevent this antibody response.